LOCUS       sequence-443133-        3918 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expression HIS-FLAG-AtUBQ11 in in bacterial cells.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 3918)
  AUTHORS   Pu ZX, Wang JL, Li YY, Liang LY, Tan YT, Wang ZH, Li BL, Guo GQ, 
            Wang L, Wu L
  TITLE     A Bacterial Platform for Studying Ubiquitination Cascades Anchored 
            by SCF-Type E3 Ubiquitin Ligases.
  JOURNAL   Biomolecules. 2024 Sep 25;14(10):1209. doi: 10.3390/biom14101209.
  PUBMED    39456142
REFERENCE   2  (bases 1 to 3918)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Dec 15, 2025 from SnapGene Server 8.0.1
            https://www.snapgene.com
COMMENT     SGRef: number: 1; type: "Journal Article"; journalName: 
            "Biomolecules."; date: "2024-09-25"; volume: "14(10):1209. doi"; 
            pages: " 10.3390/biom14101209"
COMMENT     Sequence Label: pRSFDuet-HIS-FLAG-AtUBQ11
FEATURES             Location/Qualifiers
     source          1..3918
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     46..65
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        46..64
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    65..89
                     /label=lac operator
                     /bound_moiety="lac repressor encoded by lacI"
                     /note="The lac repressor binds to the lac operator to 
                     inhibit transcription in E. coli. This inhibition can be 
                     relieved by adding lactose or 
                     isopropyl-beta-D-thiogalactopyranoside (IPTG)."
     CDS             145..162
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     CDS             172..189
                     /codon_start=1
                     /product="thrombin recognition and cleavage site"
                     /label=thrombin site
                     /translation="LVPRGS"
     CDS             214..261
                     /codon_start=1
                     /product="two tandem FLAG(R) epitope tags"
                     /label=2xFLAG
                     /translation="DYKDDDDKDYKDDDDK"
     CDS             517..561
                     /codon_start=1
                     /product="affinity and epitope tag derived from pancreatic 
                     ribonuclease A"
                     /label=S-Tag
                     /translation="KETAAAKFERQHMDS"
     primer_bind     complement(599..617)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      613..660
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     CDS             complement(890..1705)
                     /codon_start=1
                     /product="aminoglycoside phosphotransferase"
                     /label=KanR
                     /note="confers resistance to kanamycin in bacteria or G418 
                     (Geneticin(R)) in eukaryotes"
                     /translation="MSHIQRETSCSRPRLNSNMDADLYGYKWARDNVGQSGATIYRLYG
                     KPDAPELFLKHGKGSVANDVTDEMVRLNWLTEFMPLPTIKHFIRTPDDAWLLTTAIPGK
                     TAFQVLEEYPDSGENIVDALAVFLRRLHSIPVCNCPFNSDRVFRLAQAQSRMNNGLVDA
                     SDFDDERNGWPVEQVWKEMHKLLPFSPDSVVTHGDFSLDNLIFDEGKLIGCIDVGRVGI
                     ADRYQDLAILWNCLGEFSPSLQKRLFQKYGIDNPDMNKLQFHLMLDEFF"
     CDS             complement(890..1705)
                     /codon_start=1
                     /gene="aph(3')-Ia"
                     /product="aminoglycoside phosphotransferase"
                     /label=KanR
                     /note="confers resistance to kanamycin in bacteria or G418 
                     (Geneticin(R)) in eukaryotes"
                     /translation="MSHIQRETSCSRPRLNSNMDADLYGYKWARDNVGQSGATIYRLYG
                     KPDAPELFLKHGKGSVANDVTDEMVRLNWLTEFMPLPTIKHFIRTPDDAWLLTTAIPGK
                     TAFQVLEEYPDSGENIVDALAVFLRRLHSIPVCNCPFNSDRVFRLAQAQSRMNNGLVDA
                     SDFDDERNGWPVEQVWKEMHKLLPFSPDSVVTHGDFSLDNLIFDEGKLIGCIDVGRVGI
                     ADRYQDLAILWNCLGEFSPSLQKRLFQKYGIDNPDMNKLQFHLMLDEFF"
     primer_bind     1613..1632
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     promoter        complement(1706..1797)
                     /gene="bla"
                     /label=AmpR promoter
     rep_origin      complement(1813..2562)
                     /direction=LEFT
                     /label=RSF ori
                     /note="Plasmids containing the RSF 1030 origin of 
                     replication can be propagated in E. coli cells that contain
                     additional plasmids with compatible origins."
     primer_bind     complement(1823..1840)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     primer_bind     complement(2063..2082)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(2758..3840)
                     /codon_start=1
                     /gene="lacI"
                     /product="lac repressor"
                     /label=lacI
                     /note="The lac repressor binds to the lac operator to 
                     inhibit transcription in E. coli. This inhibition can be 
                     relieved by adding lactose or 
                     isopropyl-beta-D-thiogalactopyranoside (IPTG)."
                     /translation="MKPVTLYDVAEYAGVSYQTVSRVVNQASHVSAKTREKVEAAMAEL
                     NYIPNRVAQQLAGKQSLLIGVATSSLALHAPSQIVAAIKSRADQLGASVVVSMVERSGV
                     EACKAAVHNLLAQRVSGLIINYPLDDQDAIAVEAACTNVPALFLDVSDQTPINSIIFSH
                     EDGTRLGVEHLVALGHQQIALLAGPLSSVSARLRLAGWHKYLTRNQIQPIAEREGDWSA
                     MSGFQQTMQMLNEGIVPTAMLVANDQMALGAMRAITESGLRVGADISVVGYDDTEDSSC
                     YIPPLTTIKQDFRLLGQTSVDRLLQLSQGQAVKGNQLLPVSLVKRKTTLAPNTQTASPR
                     ALADSLMQLARQVSRLESGQ"
     primer_bind     3802..3821
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(3841..3918)
                     /gene="lacI"
                     /label=lacI promoter
ORIGIN
        1 cgggatctcg acgctctccc ttatgcgact cctgcattag gaaattaata cgactcacta
       61 taggggaatt gtgagcggat aacaattccc ctgtagaaat aattttgttt aactttaata
      121 aggagatata ccatgggcag cagccatcat catcatcatc acagcagcgg cctggtgccg
      181 cgcggcagcc atatggctag ctctagaatg tctgactaca aggacgacga tgacaaggac
      241 tacaaggacg acgatgacaa gggatccatg cagatctttg ttaagactct caccggaaag
      301 actatcaccc tcgaggtgga aagctctgac accatcgaca acgttaaggc caagatccag
      361 gataaggaag gtattcctcc ggatcagcag aggcttatct tcgccggaaa gcagttggag
      421 gatggccgca cgttggcgga ttacaatatc cagaaggaat ccaccctcca cttggtcctc
      481 aggctccgtg gtggttaagg taccctcgag tctggtaaag aaaccgctgc tgcgaaattt
      541 gaacgccagc acatggactc gtctactagc gcagcttaat taacctaggc tgctgccacc
      601 gctgagcaat aactagcata accccttggg gcctctaaac gggtcttgag gggttttttg
      661 ctgaaacctc aggcatttga gaagcacacg gtcacactgc ttccggtagt caataaaccg
      721 gtaaaccagc aatagacata agcggctatt taacgaccct gccctgaacc gacgacaagc
      781 tgacgaccgg gtctccgcaa gtggcacttt tcggggaaat gtgcgcggaa cccctatttg
      841 tttatttttc taaatacatt caaatatgta tccgctcatg aattaattct tagaaaaact
      901 catcgagcat caaatgaaac tgcaatttat tcatatcagg attatcaata ccatattttt
      961 gaaaaagccg tttctgtaat gaaggagaaa actcaccgag gcagttccat aggatggcaa
     1021 gatcctggta tcggtctgcg attccgactc gtccaacatc aatacaacct attaatttcc
     1081 cctcgtcaaa aataaggtta tcaagtgaga aatcaccatg agtgacgact gaatccggtg
     1141 agaatggcaa aagtttatgc atttctttcc agacttgttc aacaggccag ccattacgct
     1201 cgtcatcaaa atcactcgca tcaaccaaac cgttattcat tcgtgattgc gcctgagcga
     1261 gacgaaatac gcggtcgctg ttaaaaggac aattacaaac aggaatcgaa tgcaaccggc
     1321 gcaggaacac tgccagcgca tcaacaatat tttcacctga atcaggatat tcttctaata
     1381 cctggaatgc tgttttcccg gggatcgcag tggtgagtaa ccatgcatca tcaggagtac
     1441 ggataaaatg cttgatggtc ggaagaggca taaattccgt cagccagttt agtctgacca
     1501 tctcatctgt aacatcattg gcaacgctac ctttgccatg tttcagaaac aactctggcg
     1561 catcgggctt cccatacaat cgatagattg tcgcacctga ttgcccgaca ttatcgcgag
     1621 cccatttata cccatataaa tcagcatcca tgttggaatt taatcgcggc ctagagcaag
     1681 acgtttcccg ttgaatatgg ctcatactct tcctttttca atattattga agcatttatc
     1741 agggttattg tctcatgagc ggatacatat ttgaatgtat ttagaaaaat aaacaaatag
     1801 gcatgcagcg ctcttccgct tcctcgctca ctgactcgct acgctcggtc gttcgactgc
     1861 ggcgagcggt gtcagctcac tcaaaagcgg taatacggtt atccacagaa tcaggggata
     1921 aagccggaaa gaacatgtga gcaaaaagca aagcaccgga agaagccaac gccgcaggcg
     1981 tttttccata ggctccgccc ccctgacgag catcacaaaa atcgacgctc aagccagagg
     2041 tggcgaaacc cgacaggact ataaagatac caggcgtttc cccctggaag ctccctcgtg
     2101 cgctctcctg ttccgaccct gccgcttacc ggatacctgt ccgcctttct cccttcggga
     2161 agcgtggcgc tttctcatag ctcacgctgt tggtatctca gttcggtgta ggtcgttcgc
     2221 tccaagctgg gctgtgtgca cgaacccccc gttcagcccg accgctgcgc cttatccggt
     2281 aactatcgtc ttgagtccaa cccggtaaga cacgacttat cgccactggc agcagccatt
     2341 ggtaactgat ttagaggact ttgtcttgaa gttatgcacc tgttaaggct aaactgaaag
     2401 aacagatttt ggtgagtgcg gtcctccaac ccacttacct tggttcaaag agttggtagc
     2461 tcagcgaacc ttgagaaaac caccgttggt agcggtggtt tttctttatt tatgagatga
     2521 tgaatcaatc ggtctatcaa gtcaacgaac agctattccg ttactctaga tttcagtgca
     2581 atttatctct tcaaatgtag cacctgaagt cagccccata cgatataagt tgtaattctc
     2641 atgttagtca tgccccgcgc ccaccggaag gagctgactg ggttgaaggc tctcaagggc
     2701 atcggtcgag atcccggtgc ctaatgagtg agctaactta cattaattgc gttgcgctca
     2761 ctgcccgctt tccagtcggg aaacctgtcg tgccagctgc attaatgaat cggccaacgc
     2821 gcggggagag gcggtttgcg tattgggcgc cagggtggtt tttcttttca ccagtgagac
     2881 gggcaacagc tgattgccct tcaccgcctg gccctgagag agttgcagca agcggtccac
     2941 gctggtttgc cccagcaggc gaaaatcctg tttgatggtg gttaacggcg ggatataaca
     3001 tgagctgtct tcggtatcgt cgtatcccac taccgagatg tccgcaccaa cgcgcagccc
     3061 ggactcggta atggcgcgca ttgcgcccag cgccatctga tcgttggcaa ccagcatcgc
     3121 agtgggaacg atgccctcat tcagcatttg catggtttgt tgaaaaccgg acatggcact
     3181 ccagtcgcct tcccgttccg ctatcggctg aatttgattg cgagtgagat atttatgcca
     3241 gccagccaga cgcagacgcg ccgagacaga acttaatggg cccgctaaca gcgcgatttg
     3301 ctggtgaccc aatgcgacca gatgctccac gcccagtcgc gtaccgtctt catgggagaa
     3361 aataatactg ttgatgggtg tctggtcaga gacatcaaga aataacgccg gaacattagt
     3421 gcaggcagct tccacagcaa tggcatcctg gtcatccagc ggatagttaa tgatcagccc
     3481 actgacgcgt tgcgcgagaa gattgtgcac cgccgcttta caggcttcga cgccgcttcg
     3541 ttctaccatc gacaccacca cgctggcacc cagttgatcg gcgcgagatt taatcgccgc
     3601 gacaatttgc gacggcgcgt gcagggccag actggaggtg gcaacgccaa tcagcaacga
     3661 ctgtttgccc gccagttgtt gtgccacgcg gttgggaatg taattcagct ccgccatcgc
     3721 cgcttccact ttttcccgcg ttttcgcaga aacgtggctg gcctggttca ccacgcggga
     3781 aacggtctga taagagacac cggcatactc tgcgacatcg tataacgtta ctggtttcac
     3841 attcaccacc ctgaattgac tctcttccgg gcgctatcat gccataccgc gaaaggtttt
     3901 gcgccattcg atggtgtc
//
